The primary purpose of the present study was to compare the microbial profiles of the tongue dorsa of healthy subjects and subjects with halitosis by using culture-independent molecular methods. Our overall goal was to determine the bacterial diversity on the surface of the tongue dorsum as part of our ongoing efforts to identify all cultivable and not-yet-cultivated species of the oral cavity. Tongue dorsum scrapings were analyzed from healthy subjects with no complaints of halitosis and subjects with halitosis, defined as an organoleptic score of 2 or more and volatile sulfur compound levels greater than 200 ppb. 16S rRNA genes from DNA isolated from tongue dorsum scrapings were amplified by PCR with universally conserved bacterial primers and cloned into Escherichia coli. Typically, 50 to 100 clones were analyzed from each subject. Fifty-one strains isolated from the tongue dorsa of healthy subjects were also analyzed. Partial sequences of approximately 500 bases of cloned inserts from the 16S rRNA genes of isolates were compared with sequences of known species or phylotypes to determine species identity or closest relatives. Nearly complete sequences of about 1,500 bases were obtained for potentially novel species or phylotypes. In an analysis of approximately 750 clones, 92 different bacterial species were identified. About half of the clones were identified as phylotypes, of which 29 were novel to the tongue microbiota. Fifty-one of the 92 species or phylotypes were detected in more than one subject. Those species most associated with healthy subjects were Streptococcus salivarius, Rothia mucilaginosa, and an uncharacterized species of Eubacterium (strain FTB41). Streptococcus salivarius was the predominant species in healthy subjects, as it represented 12 to 40% of the total clones analyzed from each healthy subject. Overall, the predominant microbiota on the tongue dorsa of healthy subjects was different from that on the tongue dorsa of subjects with halitosis. Those species most associated with halitosis were Atopobium parvulum, a phylotype (clone BS095) of Dialister, Eubacterium sulci, a phylotype (clone DR034) of the uncultivated phylum TM7, Solobacterium moorei, and a phylotype (clone BW009) of Streptococcus. On the basis of our ongoing efforts to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species that colonize the oral cavity, there are now over 600 species.

Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients

Background/aim: To examine the proportions of 40 bacterial species in samples from 8 oral soft tissue surfaces and saliva in systemically healthy adult subjects and to compare these microbiotas with those of supra- and subgingival plaque.

Methods: Microbial samples were taken from 8 oral soft tissue surfaces of 225 systemically healthy subjects using a “buccal brush”. Saliva was taken by expectoration. Forty-four of these subjects provided additional supra- and subgingival plaque samples. Samples were individually evaluated for their content of 40 bacterial species using checkerboard DNA–DNA hybridization. The percentage of total DNA probe count was determined for each species, at each sample location and averaged across subjects. The significance of differences among the proportions of the 40 test species at different sample locations was sought in the 225 and 44 subjects separately using the Quade test and adjusted for multiple comparisons. Cluster analysis was performed using the proportions of the 40 species at the different sample locations using the minimum similarity coefficient and an average unweighted linkage sort. The proportions of each species were averaged across subjects in the resulting cluster groups and the significance of differences was tested using the t-test and ANOVA.

Results: Microbial profiles differed markedly among sample locations in the 225 subjects, with 34 of 40 species differing significantly. Proportions of Veillonella parvula and Prevotella melaninogenica were higher in saliva and on the lateral and dorsal surfaces of the tongue, while Streptococcus mitis and S. oralis were in significantly lower proportions in saliva and on the tongue dorsum. Cluster analysis resulted in the formation of 2 clusters with >85% similarity. Cluster 1 comprised saliva, lateral and dorsal tongue surfaces, while Cluster 2 comprised the remaining soft tissue locations. V. parvula, P. melaninogenica, Eikenella corrodens, Neisseria mucosa, Actinomyces odontolyticus, Fusobacterium periodonticum, F. nucleatum ss vincentii and Porphyromonas gingivalis were in significantly higher proportions in Cluster 1 and S. mitis, S. oralis and S. noxia were significantly higher in Cluster 2. These findings were confirmed using data from the 44 subjects providing plaque samples. The microbial profiles of supra- and subgingival plaque differed from the other sample locations, particularly in the increased proportions of the Actinomyces species. Species of different genera exhibited different proportions on the various intraoral surfaces, but even within the genus Streptococcus, there were differences in colonization patterns. S. oralis, S. mitis and S. constellatus colonized the soft tissues and saliva in higher proportions than the samples from the teeth, while the other 4 streptococcal species examined colonized the dental surfaces in proportions comparable to the soft tissue locations and saliva.

Conclusions: Proportions of bacterial species differed markedly on different intraoral surfaces. The microbiota of saliva was most similar to that of the dorsal and lateral surfaces of the tongue. The microbiotas of the soft tissues resembled each other more than the microbiotas that colonized the teeth both above and below the gingival margin.

Distribution of selected bacterial species on intraoral surfaces

Oral malodor was measured using a portable sulphide monitor in 2,672 individuals aged 18 to 64 years. In addition, dental (DMFT) and periodontal conditions (CPITN and attachment loss), dental plaque, and tongue coating status were assessed. Before clinical examination, subjects were interviewed about their oral health habits, smoking habits, and medical history. Data on volatile sulphur compounds (VSC) were analyzed by gender, age group, and time of measurement. There were no significant differences observed in the VSC between males and females in any age group. In each age group, the measured values of oral malodor were highest in the late morning group (58.6 ppb in average), followed by the late afternoon group (52.1 ppb), while lowest values were shown in the early afternoon group (39.4 ppb). Significant correlation was observed only between the VSC value and periodontal conditions and tongue coating status. The results also suggest that oral malodor might be caused mainly by tongue coating in the youn...

Correlation between volatile sulphur compounds and certain oral health measurements in the general population.

Research suggests that the tongue plays an important role in the production of oral malodor. To investigate the role of tongue surface characteristics and oral bacteria in halitosis development, the authors tested associations between odor measurements, volatile sulfur compound levels, periodontal parameters, tongue surface characteristics, presence of trypsinlike activity of organisms on the tongue and teeth and bacteriological parameters in 16 participants with complaints of oral malodor. The data indicate that the proteolytic, anaerobic flora residing on the tongue plays an essential role in the development of halitosis.

Assessing the contribution of anaerobic microflora of the tongue to oral malodor

Over the last decade, hydrogen sulfide (H2S) has emerged as an important endogenous gasotransmitter in mammalian cells and tissues. Similar to the previously characterized gasotransmitters nitric oxide and carbon monoxide, H2S is produced by various enzymatic reactions and regulates a host of physiologic and pathophysiological processes in various cells and tissues. H2S levels are decreased in a number of conditions (e.g., diabetes mellitus, ischemia, and aging) and are increased in other states (e.g., inflammation, critical illness, and cancer). Over the last decades, multiple approaches have been identified for the therapeutic exploitation of H2S, either based on H2S donation or inhibition of H2S biosynthesis. H2S donation can be achieved through the inhalation of H2S gas and/or the parenteral or enteral administration of so-called fast-releasing H2S donors (salts of H2S such as NaHS and Na2S) or slow-releasing H2S donors (GYY4137 being the prototypical compound used in hundreds of studies in vitro and in vivo). Recent work also identifies various donors with regulated H2S release profiles, including oxidant-triggered donors, pH-dependent donors, esterase-activated donors, and organelle-targeted (e.g., mitochondrial) compounds. There are also approaches where existing, clinically approved drugs of various classes (e.g., nonsteroidal anti-inflammatories) are coupled with H2S-donating groups (the most advanced compound in clinical trials is ATB-346, an H2S-donating derivative of the non-steroidal anti-inflammatory compound naproxen). For pharmacological inhibition of H2S synthesis, there are now several small molecule compounds targeting each of the three H2S-producing enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Although many of these compounds have their limitations (potency, selectivity), these molecules, especially in combination with genetic approaches, can be instrumental for the delineation of the biologic processes involving endogenous H2S production. Moreover, some of these compounds (e.g., cell-permeable prodrugs of the CBS inhibitor aminooxyacetate, or benserazide, a potentially repurposable CBS inhibitor) may serve as starting points for future clinical translation. The present article overviews the currently known H2S donors and H2S biosynthesis inhibitors, delineates their mode of action, and offers examples for their biologic effects and potential therapeutic utility.

https://pharmrev.aspetjournals.org/content/pharmrev/69/4/497.full.pdf

International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors.

Forty-one subjects with bad breath were assessed for oral malodor and periodontal status on three occasions, at intervals of approximately one week. Oral malodor was assessed by measurement of peak and steady-state volatile sulphide levels with a portable sulphide monitor and by organoleptic measurement of whole-mouth, tongue dorsum, and interproximal dental odors by two independent judges. Reproducibility of measurements, assessed by paired t tests and Kappa testing, demonstrated no significant differences between any ofthe test results from the first and second appointments. Steady-state sulphide levels were the most reproducible of all tests. The ability of the tests to detect an expected reduction of malodor following a 0.2% chlorhexidine mouthrinse regimen was investigated by comparison of test values between the second and third appointments. Following the mouthrinsing treatment, 43% reductions of peak, 47% reductions of steady-state volatile sulphide levels, and 15-58% reductions in all other measu...

Reproducibility and Sensitivity of Oral Malodor Measurements with a Portable Sulphide Monitor

Associations between oral malodor, measures of periodontal disease, and trypsin-like activity of periodontal pathogens on tongue and teeth were examined in 127 subjects. Volatile sulphur compound (VSC) measurements were made with a portable sulphide monitor; oral malodor was also estimated by organoleptic methods. Measurements repeated one week apart indicated that steady-state VSC levels (r = 0.72; P = 0.0001) and peak VSC levels (r = 0.63; P = 0.0001) were reproducible but these r values were not significantly different (P > 0.1). There was a significant correlation between tongue odor and peak VSC levels (r = 0.40; P = 0.0001) and between tongue odor and whole mouth organoleptic measures (r = 0.55; P = 0.0001). To study the effect of reducing microbial colonization on oral malodor, chlorhexidine gluconate (0.2%) rinsing was prescribed for 7 days. Reductions of VSC levels were significant for both peak (37%) and steady-state (41%) data (P = 0.0001). Anaerobic periodontal pathogens on the tongue estimated by the proportions of positive BANA tests were reduced 19% (P = 0.001) and this was concomitant with a 40% (P = 0.0001) decrease in organoleptic measurement of the tongue dorsum. Mean pH measurements of the tongue dorsum showed large reductions from 6.9 initially to 6.3 post-treatment (P = 0.0001). Subjects were divided into periodontitis/no periodontitis based on periodontal inflammation and probing depth (> or = 5 mm). Of the 37 subjects with periodontitis, 23 had oral malodor whereas 52 out of 90 periodontally healthy subjects exhibited malodor. Chi square analysis comparing halitosis in subjects with and without periodontitis showed no statistically significant association (chi 2 = 0.208; P 0.65) between these two factors although the intensity of malodor as based on VSC concentration in periodontally healthy subjects was 19% less (mean = 111 ppb) than in subjects with periodontitis (mean = 136 ppb). The odds ratio was 1.2, indicating that oral malodor was not associated with periodontitis. These data indicate that a large proportion of individuals with oral malodor are periodontally healthy and that the mucosal surface of the tongue is a major site of oral malodor production.

Relationship of Oral Malodor to Periodontitis: Evidence of Independence in Discrete Subpopulations

Background and objectives Periodontitis affects the supporting structures of the teeth as a result of the interactions between the subgingival biofilm and the host immune system. Periodontal therapy in severe forms of periodontitis often utilizes antimicrobial agents with some potential to improve host defense responses. In the present study, we investigated the in vitro effect of metronidazole (MTZ) at concentrations achievable in the periodontal pocket on PMN activation and PMN mediated killing of Porphyromonas gingivalis. Materials and methods Flow cytometry based assays were used to measure the impact of MTZ on PMN degranulation, neutrophil extracellular trap (NET) formation and myeloperoxidase (MPO) release and phagocytosis in response to the keystone oral pathogen P. gingivalis. Functional assays for PMN mediated killing of P. gingivalis and reactive oxygen species (ROS) production in PMN were also carried out. Results We demonstrate that PMNs pretreated with MTZ (2 μg/ml or 50 μg/ml) displayed enhanced killing of P. gingivalis compared to untreated PMNs. At concentrations achieved physiologically in the periodontal pocket, MTZ induced PMN surface expression of two activation markers (CD66 and CD63). MTZ did not alter P. gingivalis-induced NETosis, but suppressed P. gingivalis-induced ROS production and phagocytosis. Conclusion MTZ displays a positive interaction with PMNs to potentiate PMN mediated killing of P. gingivalis and may therefore contribute to its beneficial effects in the treatment of periodontitis initiated by P. gingivalis infections including those refractory to conventional treatment.

Metronidazole enhances killing of Porphyromonas gingivalis by human PMNs

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A. Bosy

Papers

Reproducibility and Sensitivity of Oral Malodor Measurements with a Portable Sulphide Monitor

Relationship of Oral Malodor to Periodontitis: Evidence of Independence in Discrete Subpopulations

Metronidazole enhances killing of Porphyromonas gingivalis by human PMNs

Periodontitis: Evidence of Independence in Discrete Subpopulations